Screening Of Verticillium Wilt Resistance Gene In Cotton And Functional Analysis Of NBS-LRR Resistance Gene GbaNA1

Verticillium wilt caused by Verticillium dahliae is the most destructive disease in cotton.Verticillium dahliae is a soil-borne plant pathogen that invades the water-conducting xylem vessels of susceptible plant species to cause vascular wilt disease.This results in several symptoms on leaves,including wilting,discoloration and defoliation which seriously threatened the cotton yield and fiber quality.At present,the molecular mechanisms of cotton resistance to Verticillium Wilt and the functional analysis of disease resistance genes are the the hot issues in the research of cotton resistance to Verticillium wilt.In this study,the genome sequence of diploid cotton,Gossypium raimondii,was analyzed.Highthroughput RNA-seq was used to identify the RGA genes transcriptome in a V.dahliae-resistant cultivar of G.barbadense and to screen for potential Verticillium dahliae response loci(VdRLs).One of the resistance loci VdRL08 was analyzed,by the expression pattern and virus induced genes silencing.Then the resistance genes were cloned and identified.The main results and conclusions are as follows:1.The G.raimondii genome encodes 1004 RGA genes,and most of these genes cluster in homology groups based on high levels of similarity.Interestingly,nearly half of the RGA genes occurred in 26 RGA-gene-rich clusters(Rgrcs).The homology analysis showed that sequence exchanges and tandem duplications frequently occurred within Rgrcs,and segmental duplications took place among these different Rgrcs.RNA-seq analysis showed that the RGA genes play roles in cotton defence responses,forming 26 VdRLs inside in 19 Rgrcs after being inoculated with V.dahliae.A correlation analysis found that 12 VdRLs were adjacent to the known Verticillium wilt resistance QTLs,and that 5 were rich in NB-ARC domain-containing NBS-LRR disease resistance genes.2.The Verticillium wilt resistance locus VdRL08,which is rich in NBS-LRR class resistance genes,contains 21 genes that eight of them encode NBS-LRR proteins.We investigated the gene expression pattern of these genes are different on resistance cultivar of G.barbadense and susceptible cultivar of G.hirsutum.PCR validation showed that the fragment of most encoding genes,except for five genes,in VdRL08 could successfully amply from the genomic DNA of G.barbadense.Among the seven NBS-LRR encoding genes in locus VdRL08 in G.barbadense,only silencing of GbaNA1 significantly impaired cotton Verticillium wilt resistance.Therefore,these studies suggested that the NBS-LRR type gene of GbaNA1 in VdRL08 locus should be the functional gene involving in the Verticillium wilt resistance in cotton.3.The sequence of GbaNA1 gene was 2856 bp,encoding 945 amino acids.The comparison of the genomic and cDNA sequences showed that GbaNA1 contains a single open reading frame with 2856 bp.The results of Blast showed that the gene belongs to NBS-LRR type disease resistance gene.Further evaluated the possible defense response mediates by GbaNA1 to the hormone showed that the expression pattern of GbaNA1 sensitive to ethylene(ET)and methyl jasmonic acid(Me JA).Subcellular localization showed that the protein GbaNA1 was localized to the nucleus.Compared withthe Arabisopsis wild-type Col-0 ecotype,these Gba NA1-overexpressing lines exhibited significantly enhanced-resistance to V.dahliae.GbaNA1 has the ability to recover the Verticillium wilt resistance of the mutant of its orthologue gene AT4G27220.1 in Arabidopsis.These results suggested that Gba NA1 positively regulates the Verticillium wilt resistance after interfamily transfer into the Arabidopsis.We detected the ROS activation in transgenic lines after infiltration with V.dahliae conidia suspension.As expected,the ROS activation in the GbaNA1 transgenic lines significantly enhanced than the wild-type Col-0.Similarity,overexpression of GbaNA1 in na1 mutant also displayed the higher ROS accumulation than the na1 lines.In this study,ethylene signaling-genes in transgenic lines were significantly up-regulated in GbaNA1-overexpressed in wild-type Col-0 and na1 mutant after inoculation with V.dahliae.4.We set out to isolates the GbaNA1 homolog genes in the selective cotton of G.barbadense and G.hirsutum(nine germplasm accessions of each),the open reading frames of all GbaNA1 homolog genes from the G.hirsutum accessions displayed the premature termination(stop codon at position 757-759 bp,TGA)due to the 1-bp insertion occurrence in position 713 bp.The premature termination of the protein-encoding genes in the G.hirsutum accessions caused significantly truncated protein sequence that is only consist of 252 aa.We generated the GhNA1-overexpressing lines of Arabidopsis for Verticillium wilt resistance detection.As expected,the GhNA1-overexpressing lines cannot enhance resistance to V.dahliae.The results suggested that GhNA1 from the G.hirsutum loss the Verticillium wilt resistance function.In conclusion,the RGA gene formed typical RGA-gene-rich clusters(Rgrcs)in cotton genome.26 Rgrcs in cotton genome formed in 26 VdRLs after infected by Verticillium dahliae.One of the resistance loci Vd RL08 is rich in NBS-LRR type resistance genes.The NBS-LRR type gene of Gba NA1 in Vd RL08 locus should be the functional gene involves in the Verticillium wilt resistance in cotton.Polymorphism analysis showed that GbaNA1 had a premature termination mutation in susceptible G.hirsutum,mutant gene Over-expression unable improve resistance of Arabidopsis to V.dahliae.These experimental results showed that the GbaNA1 gene in VdRL08 locus is a functional gene involving in the Verticillium wilt resistance in cotton.